First published April 16, 2012Submitted: May 31,
2011; Accepted: February 29,
2012.

Malignant progression in cancer requires populations of tumor-initiating cells
(TICs) endowed with unlimited self renewal, survival under stress, and
establishment of distant metastases. Additionally, the acquisition of invasive
properties driven by epithelial-mesenchymal transition (EMT) is critical for the
evolution of neoplastic cells into fully metastatic populations. Here, we
characterize 2 human cellular models derived from prostate and bladder cancer
cell lines to better understand the relationship between TIC and EMT programs in
local invasiveness and distant metastasis. The model tumor subpopulations that
expressed a strong epithelial gene program were enriched in highly metastatic
TICs, while a second subpopulation with stable mesenchymal traits was
impoverished in TICs. Constitutive overexpression of the transcription factor
Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene
program and suppressed their self renewal and metastatic phenotypes. Conversely,
knockdown of EMT factors in the mesenchymal-like prostate cancer cell
subpopulation caused a gain in epithelial features and properties of TICs. Both
tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like
prostate cancer subpopulation enhanced the in vitro invasiveness of the
metastatic epithelial subpopulation and, in vivo, promoted the escape of the
latter from primary implantation sites and accelerated their metastatic
colonization. Our models provide new insights into how dynamic interactions
among epithelial, self-renewal, and mesenchymal gene programs determine the
plasticity of epithelial TICs.

Introduction

There is a wealth of evidence that the acquisition of aggressive traits of cancer, or
malignant progression, can be determined both by the occurrence of genetic mutations
and by the imposition of heritable epigenetic marks on relevant genes (1). Within a tumor, these newly acquired genetic
and epigenetic events can emerge either sequentially within a single lineage or in
parallel in multiple, independent lineages (2). In either scenario of cancer cell evolution, the final outcome is the
coexistence in a given tumor of different subpopulations of tumor cells, each
endowed with particular phenotypes (intratumoral heterogeneity). There is also
evidence that transcriptional reprogramming in tumor cells can be induced in
response to nontumor environmental cues that include factors such as
TGF-β, PDGF, or EGF (3), hormones,
or hypoxic stress (4). Therefore, cancer cells
endowed with a capacity for indefinite self renewal (cancer stem cells [CSCs]), but
still retaining some capacity for differentiation, could evolve into distinct
phenotypes in response to environmental cues and to new mutations. It has been
proposed that, as in any ecological niche (5),
these subpopulations could interact among each other, either by competing for common
resources (6) or by cooperating for mutual
benefit (2, 7). These tumoral subpopulations can also interact with, and use to
their advantage, nontumoral elements, as has been convincingly shown in many models
of tumor progression and metastasis (8).

Tumor-initiating cells (TICs) constitute subpopulations of cells capable of
initiating and sustaining the growth of tumors in immunodeficient mice (9–11). In turn, TICs and CSCs share with ES and adult stem cells gene
networks that are essential for self renewal and pluripotency (12, 13). Independent of
their origin, it is still unclear whether CSCs are a population of tumor cells
endowed with irreversible self-renewal properties or whether they are subject to
dynamic influences that can affect their phenotypes (14, 15). A second process and
gene program critical for cancer progression is epithelial-mesenchymal transition
(EMT) (16–19). Whether induced by environmental cues or by other
mechanisms, EMT is driven by transcriptional factors such as
SNAI1/2, ZEB1/2, or TWIST1/2,
results in enhanced migration and invasive potentials of epithelial cells, and is
critical for the metastatic spread of epithelial tumors (16, 20, 21). In several models of cancer, the induction
of EMT potentiates self renewal and the acquisition of CSC properties (22–24). Consequently, a common notion is that EMT may be a general feature
of cancer stem or progenitor populations, associating local invasiveness with the
ability to colonize distant organs as expressions of 2 tightly interdependent gene
programs borne by the same tumor cells (15,
21). However, other models of neoplasia
have found an inverse correlation between local invasiveness and the ability of
tumor cells to colonize distant organs (25),
suggesting a dichotomy between these 2 critical features of the metastatic process,
possibly expressed by separate tumor cell subpopulations (26, 27) in which tumor
cells that display a strong epithelial phenotype are endowed with the strongest
capacity to survive in circulation and to establish distant metastases (14, 25,
28).

To better understand the relationship between CSC and EMT programs in local
invasiveness and distant metastasis, we have characterized 2 cellular models,
derived from prostate and bladder cancer cell lines, displaying a dissociation
between these 2 programs. We have found that forced induction of constitutive EMT in
subpopulations of tumor cells displaying relatively stable epithelial/TIC features
caused the suppression of major properties associated with TICs, including
anchorage-independent growth and metastatic potential. Conversely, knockdown in the
TIC-enriched epithelial subpopulations of self-renewal genes and of E-cadherin led,
in addition to an inhibition of anchorage-independent growth, to a loss of their
epithelial features, enhanced invasiveness, and an inhibition of their capacity to
colonize distant organs. These observations closely link properties of metastatic
TICs to an epithelial phenotype and gene program and suggest that, in our models,
EMT, while enabling the invasiveness of tumor cells, opposes the self-renewal gene
program that drives their local and metastatic growth.

Results

Cellular models of metastasis in which TIC and EMT properties are
dissociated. The PC-3 prostate cancer cell line was used to generate 2 distinct clonal
populations. PC-3/S cells were isolated in vitro by single-cell cloning from
luciferase-expressing PC-3 cells (29). A
second single-cell progeny, hereafter designated PC-3/Mc, was isolated from
luciferase-expressing PC-3/M cells, a PC-3 subline that had been selected in
vivo for its high metastatic potential (30). Orthotopic implantation in the ventral prostate lobe of
NOD-SCID mice of 1.0 × 105 PC-3/Mc cells quickly produced
large tumors, spreading to regional lymph nodes shortly after implantation and
to distant organs at later times (Figure 1A). In contrast, PC-3/S cells grew slowly and were not detected outside
of the implantation site for the duration of monitoring (70 days). Parental PC-3
cells displayed an intermediate behavior in local growth rate and in the
dissemination to regional lymph nodes and distant sites (Figure 1A). Intramuscular (i.m.) grafting
corroborated the remarkable differences in tumorigenicity between these 2 cell
subpopulations (Figure 1B). Grafting of
limited numbers of PC-3/Mc cells yielded robust tumor growth (Figure 1C), and they could be serially transplanted
in immunodeficient mice, maintaining or gaining their efficiency for local
growth upon successive transplantations (Supplemental Figure 1A; supplemental
material available online with this article; doi:
10.1172/JCI59218DS1). They also readily colonized lungs and bones
after i.v. (Figure 1D) or intracardiac
(i.c.) (Figure 1E and Supplemental Figure
1B) injection, suggesting enrichment in metastasis-prone TICs. In contrast,
PC-3/S cells did not grow detectable colonies in lungs or bones after i.v. or
i.c. injection at any time during monitoring (Figure 1, D and E). Therefore, the PC-3/Mc and PC-3/S
subpopulations of PC-3 prostate cancer cells display highly contrasting
phenotypes, with PC-3/Mc cells enriched in serially transplantable TICs with
high metastatic potential.

In vitro, PC-3/Mc cells grew much faster and had 1.5-fold more cells in the S
phase of the cell cycle than PC-3/S cells (Figure 2A and Supplemental Figure 2A). Likewise, PC-3/Mc cells readily
formed large spheroids under nonadherent growth conditions (Figure 2B and Supplemental Figure 2B) and maintained
this capacity upon serial plating (Supplemental Figure 2C), whereas PC-3/S cells
showed limited anchorage-independent growth (Figure 2B and Supplemental Figure 2B). Contrary to our
expectations, PC-3/Mc cells were barely invasive in Matrigel-Boyden chamber
assays, while PC-3/S cells were highly invasive and motile (Figure 2, C and F). Thus, the in vitro invasiveness
and motility of PC-3/Mc and PC-3/S cells are inversely correlated to their in
vitro spheroid-forming and proliferative potentials. This suggests a dichotomy
in these cells between 2 processes that determine the capacity of tumor cells to
metastasize, namely the capacities to grow serially transplantable tumors in
vivo and spheroids in vitro and to invade through extracellular matrix in vitro.

Gene profiling revealed a striking divergence in transcriptional programs between
these 2 subpopulations derived from a common parental cell line (Supplemental
Table 1 and Supplemental Figure 3). PC-3/Mc cells expressed an epithelial gene
program, including E-cadherin (CDH1), EpCAM
(TACSTD1), and desmoplakin (DSP), and also
genes associated with pluripotency and self renewal (31, 32), including
KLF4, MYC, SOX2,
KLF9, and LIN28A (Figure 2, D, E, G, and Supplemental Figure 3A). Gene
set enrichment analysis revealed that PC-3/Mc cells have very active DNA repair,
DNA replication, and mitotic transition and checkpoint gene networks
(Supplemental Table 2 and Supplemental Figure 3, A and B). Importantly, PC-3/Mc
cells were strongly enriched in an ES cell–like module (ESC-like
module) shown to be highly active in epithelial cancers associated with
metastasis and death (13), with 265 of
the 335 genes of this module overrepresented in PC-3/Mc cells and also in a
MYC gene module (33)
and ES1 and ES2 gene sets (ref. 12,
Figure 2H, and Supplemental Table 4). This
supports the conclusion that PC-3/Mc cells, which have a high potential for
anchorage-independent and metastatic growth but are poorly invasive in vitro,
display both an epithelial phenotype and a very active self-renewal/pluripotency
gene program. In contrast, PC-3/S cells expressed high levels of many
mesenchymal markers (e.g., VIM, SPARC, and
FN1) and genes linked to EMT, such as
TWIST2, SNAI2, ZEB1, and
RUNX2 (Figure 2, D and
E, and Supplemental Figure 3, C and D). Of interest, PC-3/S cells expressed many
genes for chemokines and inflammatory cytokines and their receptors at levels
much higher than those of PC-3/Mc cells (Supplemental Tables 3 and 5 and
Supplemental Figure 3, C and D), suggesting that this subpopulation has engaged
a proinflammatory program similar to that induced in cells under stress or in
presenescent states (34, 35). Intriguingly, PC-3/Mc cells expressed
higher levels than PC-3/S cells of the EMT factor SNAI1. The endogenous SNAI1
protein showed a correct nuclear localization in PC-3/Mc cells, clearly visible
when allowed to accumulate upon treatment with the GSK3 inhibitor LiCl or the
proteasome inhibitor MG132 (Supplemental Figure 4A). However, knockdown of
endogenous SNAI1 in PC-3/Mc cells did not significantly alter
the levels of expression of E-cadherin or other epithelial markers, suggesting a
defect in the function of endogenous SNAI1 in these cells
(Supplemental Figure 4, B and C), possibly explaining why the expression of this
factor in PC-3/Mc cells still allows the expression of high levels of E-cadherin
and a strong epithelial phenotype.

Analysis of histone marks associated with relevant promoters supported the
transcriptional basis for these divergent expression profiles (Supplemental
Figure 5). Thus, the SOX2 and E-cadherin
(CDH1) promoters were enriched in acetylated histone H4 in
PC-3/Mc but not in PC-3/S and were more enriched in the H3K27me3
repressive mark in PC-3/S. Conversely, the promoters of the mesenchymal genes
TWIST2 and RUNX2 were enriched in
acetylated histones H3 and H4 only in PC-3/S cells and were impoverished in
H3K27me3 in either cell type.

We next determined whether the epithelial-aggressive versus
mesenchymal-nonaggressive dichotomy observed in our PC-3 prostate cancer cell
line subpopulations applied to other models for which epithelial tumor cell
subpopulations with distinct potentials for growth and metastasis had been
characterized. We chose a cellular model derived from the human bladder cancer
cell line T24 (TSU-Pr1 and TSU-Pr1-B2) (36, 37). It had been
previously shown that the more epithelial TSU-Pr1-B2 cells are more tumorigenic
and metastatic than the more mesenchymal TSU-Pr1 subpopulation and can colonize
bones after i.c. inoculation in immunodeficient mice (36, 37). We
confirmed that TSU-Pr1-B2 cells display features of epithelial cells when
compared with the more mesenchymal TSU-Pr1 subpopulation: higher E-cadherin and
desmoplakin expression levels and lower levels of fibronectin and EMT factors
(SNAI2, ZEB1, ZEB2)
(Supplemental Figure 6, A–C). Compared with the mesenchymal-like
TSU-Pr1 cells, epithelial TSU-Pr1-B2 cells expressed higher levels of the
pluripotency factors SOX2, LIN28A,
NANOG, and KLF9 (Supplemental Figure 6,
A–C). Functionally, the more epithelial TSU-Pr1-B2 cells formed
significantly more and larger spheroids than the mesenchymal-like TSU-Pr1 cells,
but were significantly less invasive in vitro (Supplemental Figure 6, D and E).
Upon i.c. injection in immunocompromised mice, the more epithelial TSU-Pr1-B2
cells established metastases to the bones and other organs significantly more
efficiently and at earlier times than the more mesenchymal TSU-Pr1 cells
(Supplemental Figure 6F). Thus, the TSU-Pr1 and TSU-Pr1-B2 bladder cancer dual
cell model share gene expression and functional features with the PC-3 prostate
cancer model described above.

The maintenance of critical properties of TICs is associated with an
epithelial gene program in prostate and bladder cancer cells. We next determined whether selection from parental PC-3 cells of a subpopulation
with epithelial features could enrich for cells with higher
anchorage-independent growth potential. About 11% of parental PC-3 cells
expressed high levels of E-cadherin (Figure 3A). Sorting of cells at the top 1% level of CDH1 expression
selected for a subpopulation (PC-3/CDH1hi) that expressed higher
levels than parental or CDH1lo PC-3 cells of epithelial markers such
as CDH1 and EPCAM, and also
LIN28A, SOX2, MYC,
POU5F1/OCT4, and KLF4 (Figure 3, B–D). PC-3/CDH1hi
cells formed significantly more spheroids (Figure 3E) and were less invasive (Figure 3F) than parental or CDH1lo PC-3 cells. Additional
cell-surface phenotyping showed that most PC-3/Mc cells were
CD44hiCD24hiCD71hiCD40lo, while
most PC-3/S cells were
CD44hiCD24loCD71loCD40hi
(Supplemental Table 6). Although several studies have associated prostate and
breast CSCs with a CD44hiCD24lo profile (10, 38, 39), other reports have
found that CSCs and aggressive tumors can express high levels of CD24 (12, 40, 41). Transferrin receptor
(CD71) is expressed in actively cycling compartments in different tissues (42). Therefore, PC-3 cells enriched for an
epithelial phenotype show a stronger expression of self-renewal/pluripotency
gene networks.

E-cadherin–positive PC-3 cells show an enhanced
anchorage-independent growth and a stronger expression of a self-renewal
gene program relative to parental or E-cadherin–negative
cells. (A) Over 99% of PC-3/Mc cells were positive, and 0.3% of PC-3/S
cells were positive for surface E-cadherin. A minor fraction (11.5%) of
parental PC-3 prostate cancer cells expressed cell-surface E-cadherin. The
circle on the right panel indicates the 1% sorted population with the
highest CDH1 expression (PC-3/CDH1hi). (B) The bulk
of parental PC-3 cells displayed a spindled morphology and low levels of
membrane-bound E-cadherin. Most PC-3/CDH1hi cells displayed a
round morphology and a strong expression of membrane-bound E-cadherin. Scale
bars: 20 μm. (C) PC-3/CDH1hi cells
expressed higher levels of MYC and SOX2 and lower levels of the mesenchymal
markers fibronectin or ZEB1 than PC-3/S or PC-3/CDH1lo cells, as
determined by Western blotting. (D) PC-3/CDH1hi
cells expressed self-renewal/ pluripotency genes at levels significantly
higher than parental PC-3 cells, as determined by real-time qPCR. Relative
transcript levels are represented as the log10 of ratios between
the 2 subpopulations of their
2–ΔΔCp real-time PCR
values. (E) PC-3/CDH1hi cells grew more spheroids
than E-cadherin–negative (PC-3/CDH1lo) or parental
PC-3 cells. For comparison, the spheroid growth of PC-3/Mc and PC-3/S cells
is also illustrated. (F) PC-3/CDH1hi cells were less
invasive in Transwell-Matrigel assays than PC-3/CDH1lo or
parental PC-3 cells. For comparison, the invasiveness of PC-3/Mc and PC-3/S
cells is also illustrated. Results are expressed as mean ± SEM.
*P < 0.05; **P < 0.01.

To explore whether maintenance of an epithelial gene program is important for the
properties of PC-3/Mc or TSU-Pr1-B2 cells, we induced a mesenchymal program by
transduction and overexpression of the EMT transcription factors
Snai1, Twist1, and TWIST2
(Supplemental Figure 7). This caused, in addition to the expected changes to
more fibroblastoid morphologies (Figure 4A
and Supplemental Figure 8A) and enhanced invasiveness (Figure 4B and Supplemental Figure 8B), a reduced
formation of spheroids (Figure 4C and
Supplemental Figure 8C), in particular in response to the overexpression of
Snai1. These phenotypic changes were accompanied with a
downregulation of the self-renewal/pluripotency factors KLF4,
SOX2, and MYC in addition to a
downregulation of the epithelial markers E-cadherin, EpCAM, and desmoplakin and
upregulation of the mesenchymal markers fibronectin and SPARC (Figure 4, D–F, and Supplemental Figure
8, D–F). As expected (43),
the constitutive overexpression of exogenous SNAI1 in PC-3/Mc
cells strongly suppressed the expression of endogenous Snai1
transcripts (Figure 4F and Supplemental
Figure 8F). The switch in transcriptional programs caused by high levels of
exogenous Snai1 was accompanied by an enrichment of the
repressive histone mark H3K27me3 and depletion of the active
transcription marks acetylated histones H3 and H4 at the promoters of
SOX2 and CDH1 (Supplemental Figure 9). In
addition, the overexpression of Snai1 in PC-3/Mc cells caused a
decreased growth rate (Supplemental Figure 10A) and decreased the number of
cells in the S phase of the cell cycle (Supplemental Figure 10B). In vivo,
overexpression of Snai1 in PC-3/Mc cells led to a significant
inhibition of local growth upon orthotopic (Figure 5A) or i.m. implantation (Figure 5B) as well as inhibition of their capacity to spread to regional
lymph nodes and distant sites (Figure 5A)
and to colonize lungs (Figure 5C) or bones
(Figure 5D). Likewise, constitutive
overexpression of Snai1 in TSU-Pr1-B2 bladder cancer cells led
to a marked suppression of their potential for distant organ colonization
(Supplemental Figure 8G).

Overexpression of Snai1 in PC-3/Mc cells induces EMT and
suppresses anchorage-independent growth and the expression of a self-renewal
gene program. (A) Overexpression of Snai1,
Twist1, or TWIST2 in PC-3/Mc cells
induced a fibroblastoid morphology and a downregulation of
membrane-associated E-cadherin. Cells were transduced with retroviruses for
the expression of mouse Snai1 or Twist1 or
human TWIST2. Controls were PC-3/Mc cells transduced with
pBABE and selected for puromycin resistance. Scale bars: 20 μm.
(B) Overexpression of Snai1 strongly
induced the invasiveness of PC-3/Mc cells, with a moderate effect by
Twist1 or TWIST2. (C)
Overexpression of Snai1 strongly inhibited spheroid growth
by PC-3/Mc cells, with a moderate effect by TWIST2.
(D) Overexpression of Snai1 in PC-3/Mc
cells caused a strong downregulation of cell-surface E-cadherin, with a
moderate effect by Twist1 or TWIST2, as
determined by flow cytometry. (E) Overexpression of
Snai1 in PC-3/Mc cells induced a downregulation of
E-cadherin and EpCAM, a modest downregulation of SOX2 and MYC, and an
upregulation of fibronectin and SPARC, as determined by Western blotting.
Overexpression of Twist1 or TWIST2 induced
a moderate downregulation of E-cadherin. (F) Overexpression of
Snai1 and, more moderately, Twist1 or
TWIST2, caused a downregulation of self-renewal and
epithelial genes and an upregulation of mesenchymal genes. Relative
transcript levels are represented as the log10 of ratios between
experimental and control cells of their
2–ΔΔCp real-time PCR
values. The levels of SNAI1 correspond to the endogenous,
human transcripts, downregulated by overexpression of the exogenous (mouse)
Snai1. Asterisk in F indicates that values
for ectopic TWIST2 are off scale. Results are expressed as
mean ± SEM. *P < 0.05;
**P < 0.01; ***P <
0.001.

In reciprocal experiments, the mesenchymal-like PC-3/S tumor cells were
manipulated to reduce the levels of EMT factors. Knockdown in these cells of
SNAI1, ZEB1, or TWIST2 or
a triple knockdown (SNAI1, ZEB1, and
TWIST2) (Supplemental Figure 11) caused a loss of their
fibroblastoid morphology (Figure 6A), an
upregulation of E-cadherin (Figure 6, A, B,
and E), decreased invasiveness (Figure 6C),
and enhanced spheroid formation (Figure 6D), features that were more evident with the triple knockdown. This
phenotypic switch was accompanied with the upregulation of genes characteristic
of epithelial and self-renewal programs (Figure 6E).

Knockdown of EMT transcription factors in mesenchymal-like PC-3/S cells
causes a gain in anchorage-independent growth and the expression of a
self-renewal gene network. (A). Knockdown of SNAI1, ZEB1,
TWIST2, or a triple SZT knockdown in PC-3/S cells was
associated with fewer cells with fibroblastoid morphologies and a gain in
the expression of E-cadherin, most evident in ZEB1
knockdowns (single or triple SZT). Scale bars: 20 μm.
(B) Knockdown in mesenchymal-like PC-3/S cells of
SNAI1, ZEB1, or a triple SZT knockdown
caused an upregulation of E-cadherin, as determined by Western blotting,
with the strongest effect observed in the triple knockdown. (C)
Knockdown of SNAI1, ZEB1,
TWIST2, or a triple SZT knockdown caused a diminished
invasive capacity of PC-3/S cells in Transwell-Matrigel assays, with the
triple SZT knockdown showing the strongest effects. (D)
Knockdown of SNAI1, ZEB1,
TWIST2, or a triple SZT knockdown caused a gain in the
capacity of PC-3/S cells to grow spheroids, with the triple knockdown
showing the strongest effects. (E) Knockdown of
SNAI1, ZEB1, TWIST2,
or a triple SZT knockdown in mesenchymal-like PC-3/S cells caused an
upregulation of the epithelial genes CDH1,
EPCAM, and DSP and of the
self-renewal/ pluripotency genes LIN28,
SOX2, MYC, and KLF4,
most evident for the triple SZT knockdown. Real-time RT-PCR values,
determined by the ΔΔCp method, are represented as a
heat map with pseudocoloring ranging from green (underexpressed relative to
values in control PC-3/S cell) to red (overexpressed relative to control PC-
3/S cells). Controls were puromycin-selected PC-3/S cells bearing control
pLK0-scrambled lentiviral vector. Results are expressed as mean
± SEM. *P < 0.05; **P
< 0.01.

In support of the importance of an epithelial phenotype in the maintenance of
properties of self renewal and metastatic potential, knockdown of E-cadherin in
PC-3/Mc cells (Figure 7, A, D, and E)
caused, in addition to the expected enhanced invasiveness (Figure 7B), a significant reduction in their
capacity to form spheroids (Figure 7C) and
to colonize lungs in NOD-SCID mice (Figure 7F). Knockdown of E-cadherin in PC-3/Mc cells was accompanied with a
modest but relatively broad downregulation of self-renewal/pluripotency
transcription factors, including SOX2, KLF4,
and MYC, and the upregulation of several mesenchymal genes,
such as FN1 and ZEB2 (Figure 7, A and D), suggesting that the expression
of E-cadherin may play an active role in the maintenance of a epithelial gene
program.

Conversely, overexpression of exogenous E-cadherin in PC-3/S cells, which do not
express this epithelial marker under standard growth conditions (Figure 7G), caused a strong inhibition of
invasiveness (Figure 7H) and a striking
gain in the capacity of cells to form spheroids (Figure 7I). Upon i.m. implantation in immunocompromised mice, and
consistent with their in vitro phenotypes, PC-3/S-CDH1 cells grew tumors at
significantly faster rates than control cells (Figure 7J). This phenotypic switch was accompanied with a modest
upregulation of self-renewal/pluripotency factors and an inhibition of the
mesenchymal-like gene profile characteristic of PC-3/S cells (Figure 7K).

The above results suggest a tight association between the expression of an
epithelial gene program and the maintenance of a self-renewal gene program and
properties of TICs, as well as an inhibition of the latter properties by
induction of a mesenchymal gene program. To further explore the relationship
between the self-renewal gene network and the growth properties of PC-3/Mc
cells, we knocked down SOX2, KLF4, or
MYC, or all 3 transcripts in these cells (Supplemental
Figure 12). This caused a downregulation of E-cadherin and other epithelial
markers (Figure 8, A–C), a
decrease in the formation of spheroids (Figure 8D), and enhanced invasiveness (Figure 8E), changes that were most evident in cells with a triple knockdown
for all 3 self-renewal/pluripotency factors. In vivo, knockdown of
SOX2 was sufficient to inhibit the local growth of PC-3/Mc
cells (Figure 8F) and their lung
colonization (Figure 8G). Knockdown of
SOX2 in TSU-Pr1-B2 bladder cancer cells also resulted in a
downregulation of E-cadherin (Supplemental Figure 13, A and B), loss of
spheroid-forming potential (Supplemental Figure 13C), gain in invasiveness
(Supplemental Figure 13D), and a strong inhibition of distant organ colonization
(Supplemental Figure 13E).

In a reciprocal approach, the transduction and overexpression of
SOX2 in PC-3/S cells caused the upregulation of E-cadherin
and downregulation of fibronectin (Supplemental Figure 14A) and enhanced the
formation of spheroids (Supplemental Figure 14B) concomitant with an inhibition
of invasiveness (Supplemental Figure 14C), and a strong enhancement of
tumorigenicity upon i.m. implantation (Supplemental Figure 14D).

Taken together, these observations reinforce the notion that expression of an
epithelial gene program and phenotype is critical for the maintenance of a
self-renewal gene program and more aggressive attributes of these tumor
cells.

A cell subpopulation enriched in TICs cooperates with a subpopulation with
traits of stable EMT for enhanced in vitro invasiveness and in vivo organ
colonization. The above results suggest that tumor cells with strong epithelial phenotypes and
low autonomous (in vitro) invasive potential display strong metastatic
potential. However, in order to develop distant metastases, tumor cells must
first breach local barriers that contain them within their primary site. That
the highly metastatic PC-3/Mc cells are poorly invasive in vitro may contradict
this principle unless they become invasive under certain conditions. Indeed,
shortly after i.m. grafting in immunodeficient mice, PC-3/Mc cells downregulated
E-cadherin and upregulated fibronectin (Figure 9A), suggesting that murine factors may induce EMT in these cells in
vivo. On the other hand, tumors and lung colonies formed by PC-3/Mc cells in
NOD-SCID mice coexpressed SOX2 and E-cadherin (Figure 9B), suggesting that PC-3/Mc cells, when implanted alone in
vivo, may escape their primary implantation sites aided by EMT induced by murine
factors and that, after leaving their primary site, they may revert to an
epithelial phenotype in order to grow distant metastases.

Downregulation of E-cadherin from PC-3/Mc cells at primary implantation
sites and maintenance of its expression in lung metastasis. (A) Downregulation of E-cadherin and upregulation of fibronectin
in PC-3/Mc cells after implantation in NOD-SCID mice. Seven days after i.m.
implantation, PC-3/Mc cells, homogeneously positive for E-cadherin and
negative for fibronectin in culture prior to implantation, become
heterogeneous for expression of membrane-associated E-cadherin, as
determined by immunohistochemistry (left panel), and downregulate E-cadherin
and upregulate fibronectin, as determined by Western blotting (right panel).
Lanes separated by the white lines were run on the same gel but were
noncontiguous. Scale bar: 100 μm. (B) PC-3/Mc cells
that had metastasized to lungs after i.v. injection were largely positive
for nuclear SOX2 and membrane-associated E-cadherin, as determined by
immunohistochemistry. Scale bars: 100 μm.

In addition, the mirror-image phenotypes of subpopulations with either epithelial
or mesenchymal phenotypes in our prostate and bladder cancer models raise the
question of whether diverse populations isolated from a common parental tumor
cell line might interact with each other in order to compound a collective
behavior that has an impact on the tumor’s potential for local
invasiveness or establishment of distant metastases (2, 6, 7, 26). Upon coculture with PC-3/S cells, PC-3/Mc cells became invasive
(Figure 10A), suggesting a cooperation
between these 2 subpopulations in order to facilitate the local invasiveness of
the more epithelial tumor cell subpopulations, which display a poor autonomous
invasive potential in vitro. In addition, we found that PC-3/Mc cells that had
been cocultured with PC-3/S cells were still invasive after separation from
PC-3/S cells by FACS (Supplemental Figure 15), a phenotypic change that was
maintained for at least 7 days after coculture (Figure 10B) and was reversible, following a time-dependent decline
after separation from PC-3/S cells (Figure 10, B and F). Coculture of PC-3/Mc cells with NIH3T3 fibroblasts
also stimulated their invasiveness (Supplemental Figure 16), suggesting that the
invasiveness of epithelial PC-3/Mc can be enhanced by exposure to both tumoral
and nontumoral mesenchymal cell types. We further found that conditioned medium
(CM) from PC-3/S cells markedly induced the invasiveness of PC-3/Mc cells
(Figure 10C), suggesting that diffusible
factors play a significant role in the stimulation of invasiveness of PC-3/Mc
cells induced by PC-3/S cells. Moreover, PC-3/Mc cells that had been cocultured
with PC-3/S cells not only gained in invasive potential, but were inhibited in
their anchorage-independent growth potential (Figure 10D), reminiscent of the inhibition of self renewal and
anchorage-independent growth by EMT observed in the experiments described above.

PC-3/S cells enhance the invasiveness of PC-3/Mc cells. (A) Coculture with PC-3/S cells induced the invasiveness of
PC-3/Mc cells. Green-labeled PC-3/Mc cells were cocultured with red-labeled
PC-3/S cells on Transwell units and green or red fluorescent invading cells
scored by flow cytometry. Controls were green-labeled PC-3/Mc cells
cocultured with unlabeled PC-3/Mc cells. (B) The enhanced
invasiveness of PC-3/Mc cells was maintained for several days after
coculture with PC-3/S cells. GFP-labeled PC-3/Mc cells were cocultured for
48 hours with red-labeled PC-3/S cells, sorted, and assayed for invasiveness
either immediately or 7 days later. (C) Diffusible factors
secreted by PC-3/S cells enhanced the invasiveness of PC-3/Mc cells. PC-3/Mc
cells were exposed for 48 hours to CM from PC-3/S cells (S-CM) and assayed
for invasiveness. (D) Coculture with PC-3/S cells inhibited the
spheroid growth of PC-3/Mc cells. GFP-expressing PC-3/Mc cells and
RFP-expressing PC-3/S cells were cocultured and scored for spheroids after
14 days. (E) Coculture of PC-3/Mc cells with PC-3/S cells
induced a downregulation of E-cadherin and an upregulation of fibronectin.
Green-labeled PC-3/Mc cells and red-labeled PC-3/S cells were cocultured for
48 hours, sorted, and analyzed by Western blotting. (F) PC-3/Mc
cells cocultured with PC-3/S cells shifted their transcriptional programs
following a time-dependent reversion after coculture. Green-labeled PC-3/Mc
cells were cocultured with red-labeled PC-3/S cells for 48 or 96 hours,
sorted, and analyzed either immediately (day 0) or 7 days after sorting (day
7). Relative qPCR transcript levels are represented as a heat map (green,
underexpressed relative to control PC-3/Mc; red, overexpressed). Results are
expressed as mean ± SEM. *P < 0.05;
**P < 0.01; ***P <
0.001.

The observed phenotypic switch was accompanied with a downregulation of the
epithelial genes CDH1, EPCAM, and
DSP and the self-renewal/pluripotency genes
SOX2, OCT4, KLF4, and
LIN28A along with upregulation of the mesenchymal genes
FN1, SPARC, TWIST2, and
RUNX2, which also declined with time after coculture
(Figure 10, E and F). Changes in histone
marks at relevant promoters support an epigenetic basis for this gene program
switch (Supplemental Figure 17) and may help explain the persistence of the
invasive program of PC-3/Mc cells 7 or more days after their coculture and
separation from PC-3/S cells (Figure 10, B
and F). These observations suggest that the escape of epithelial/TIC
subpopulations from local environments may follow not only passive mechanisms
through the action of stromal components and mesenchymal tumor cells (25), but also an active mechanism through
transient EMT of epithelial tumor subpopulations induced by mesenchymal tumor
subpopulations.

In vivo, PC-3/Mc cells coinjected with PC-3/S cells grew at significantly slower
rates than PC-3/Mc cells alone upon orthotopic (Figure 11A) or i.m. (Figure 11B) implantation, but metastasized to regional lymph nodes after
orthotopic implantation at earlier times than control PC-3/Mc cells alone
(Figure 11A). In these experiments,
Renilla luciferase–expressing PC-3/S cells were
transiently detected outside of the site of orthotopic implantation at early
times after orthotopic implantation (Figure 11A), but ceased to be detected after more prolonged monitoring both
at the site of implantation or at distant sites (Figure 11A), consistent with the highly invasive but poorly
metastatic properties of these cells. These results are also consistent with the
in vitro observations, described above, of reduced growth of PC-3/Mc cells when
cocultured with PC-3/S cells, along with an increased capacity to escape from
implantation sites, followed by a subsequent restoration of their growth
potential at distant sites where PC-3/S cells are no longer present. Upon i.v.
injection, PC-3/Mc cells coinjected with PC-3/S cells colonized lungs at earlier
times than PC-3/Mc cells alone (Figure 11C), suggesting that interaction with PC-3/S cells also rendered them
more efficient at extravasation. Indeed, upon i.c. inoculation, PC-3/Mc cells
coinjected with PC-3/S cells were not more efficient than PC-3/Mc cells injected
alone at colonizing bones (Figure 11D),
where the permeable sinusoidal capillary system of bone marrow represents a much
weaker barrier to extravasation than the capillaries in the lungs or other
organs (44). Additionally, PC-3/Mc cells
coinjected i.c. with PC-3/S cells colonized adrenal glands (Supplemental Table
7), which they never colonized when injected alone. Coinjection of PC-3/Mc cells
expressing green fluorescent protein together with PC-3/S cells expressing red
fluorescent protein (RFP) and Renilla luciferase allowed us to
identify the cells of origin of the tumors that developed in distant organs.
Such tumors contained only green fluorescence but not red fluorescence
— or Renilla luciferase–expressing
cells, indicating that only PC-3/Mc cells contributed to distant organ
colonization (Figure 11A and Figure 12).

Metastases formed after joint injection of PC-3/Mc and PC-3/S cells
contain exclusively epithelial PC-3/Mc cells. (A) Only PC-3/Mc cells, but not PC-3/S cells, colonized lungs
after joint i.v. injection. PC-3/S, but not PC-3/Mc, cells also expressed
Renilla luciferase. Firefly luciferase, but not
Renilla, signal was detected in lung tumors. In
parallel, GFP (expressed by GFP-PC-3/Mc cells) or RFP (expressed by
RFP-PC-3/S cells) was visualized microscopically. Only GFP signal, but not
RFP signal, was detected in lung tumors. (B) Only PC-3/Mc
cells, but not PC-3/S cells, colonize adrenal glands after i.c. joint
inoculation. Thirty-three days after inoculation, mice were sacrificed and
adrenal metastatic tumors frozen and processed for fluorescent visualization
of GFP or RFP and for immunofluorescent detection of firefly luciferase (as
a marker common to both cell types). Samples were counterstained for nuclei
with DAPI. Only GFP signal, but not RFP signal, was detected in adrenal
metastases. Scale bars: 50 μm.

These results suggest that, in vivo, while the mesenchymal-like PC-3/S tumor
cells can escape local tumor sites but lack metastatic potential, their presence
facilitates the escape of the more epithelial PC-3/Mc cells from local tumor
sites in order to establish distant metastases.

Expression of a self-renewal gene network active in PC-3/Mc cells is
associated with more advanced stages of prostate cancer. The above observations suggest that more aggressive tumors contain larger
representations of epithelial tumor cells with high self-renewal potential. To
determine whether the self-renewal gene network active in the more epithelial
PC-3/Mc subpopulation is associated with aggressive prostate cancers, we
extracted a subset of the ESC gene set (12) that most significantly discriminated PC-3/Mc from PC-3/S cells
(designated M gene set; Supplemental Table 8) and interrogated it for its
enrichment in an expression data set for 150 samples from prostate cancer
patients. (45). This analysis showed that
the M gene set is indeed significantly enriched in metastatic relative to
primary prostate cancer samples and also in primary tumor samples from more
advanced stages (T3 and T4 vs. T1 and T2) (Figure 13, A and B).

Expression of a self-renewal gene network active in PC-3/Mc cells is
associated with more advanced stages of prostate cancer. (A) GSEA on an expression data set for 150 prostate cancer
samples (45) showing a significant
enrichment of the M geneset (genes of the ESC module [ref. 13] enriched in PC-3/Mc cells) in
metastases relative to primary tumors, and in T3 and T4 stage primary tumors
relative to T1 and T2 stage primary tumors. Pearson’s
correlation was applied to determine linear relationships between gene
profiles and 3 phenotypes (class 1: metastatic; class 2: T3 and T4 stage
primary; class 3: T1 and T2 stage primary) taken as continuous variables.
(B). Heat map illustrating the relative expression levels
for the 70 genes of the M gene set. Samples are ordered as primary tumors
with stages T1 or T2, stages T3 or T4, or metastases (M). (C)
Ninety-four cases of prostate cancer were analyzed for SOX2 expression by
immunohistochemistry. Positive cases contained at least 10% of cells with
nuclear SOX2 staining. *P < 0.05, between the
frequencies of SOX2-positive cases in stages T2A and T2C versus and stage
T3A and T3B tumors. (D) In some lymph node metastases, but in
none of the 94 primary tumors, all visible tumor cells were strongly
positive for nuclear SOX2, and stronger SOX2 expression correlated with
stronger E-cadherin expression. Right: a second metastatic sample with a
more heterogeneous and weaker nuclear SOX2 staining of tumor cells displays
weaker membrane E-cadherin staining. Scale bars: 100 μm.

To determine whether these observations could be applied in in situ
histopathological analyses, we studied the expression of SOX2 as a potential
indicator of self-renewing populations by immunohistochemistry on samples from
primary and metastatic prostate cancer (Supplemental Table 9). The results
revealed a significantly higher frequency of SOX2-positive samples in stage T3
than in stage T2 tumors (Figure 13C).
Strikingly, in several SOX2-positive metastases, all tumor cells expressed SOX2
with a strong nuclear staining. Such strong and homogeneous expression of SOX2
was not observed in any of the primary tumors studied. In addition, those
metastatic samples with the strongest expression of SOX2 showed the strongest
staining for E-cadherin (Figure 13D). This
suggests that some prostate cancer metastases are enriched in tumor cells with
active self-renewal programs expressing high levels of SOX2 and E-cadherin,
consistent with the finding by Tsuji et al. that cells that colonize to organs
are non-EMT cells (25) and also with
studies describing a stronger expression of E-cadherin in metastatic tumors
(46, 47).

Discussion

A major focus of study of the metastasis problem is understanding the mechanisms by
which tumor cells escape the local environment and colonize distant organs (19, 20,
48). It has been proposed that the
engagement of an EMT program simultaneously leads, through mechanisms not yet
elucidated, to the acquisition of a self-renewal program (24, 48), endowing tumor
cells not only with the capacity to invade and migrate through tissues, but also to
survive in the circulation and form colonies in distant organs. The latter
hypothesis is largely based on experiments in which the expression of transcription
factors that direct the expression of EMT programs is manipulated for overexpression
or silencing in relatively heterogeneous populations of tumor cells and on the study
of their capacity to form tumors, invade local tissues, and establish metastases in
immunocompromised mice.

Here, we have studied clonal populations derived from the PC-3 prostate cancer and
the TSU-Pr1 bladder cancer cell lines displaying relatively stable and contrasting
phenotypes, namely cells with a strong epithelial phenotype (PC-3/Mc and TSU-Pr1-B2)
and a more mesenchymal phenotype (PC-3/S and TSU-Pr1), as determined by the
expression of genes characteristic of either program. Our analysis shows that the
subpopulations with the stronger epithelial phenotypes display clearly enhanced
capacities to form spheroids in culture and to colonize lungs and bone, compared
with the tumor subpopulations with stable mesenchymal-like phenotypes, despite the
fact that the latter are more invasive through extracellular matrix in in vitro
assays.

In order to further explore the hypothesis that a strong self-renewal and metastatic
phenotype requires the maintenance of an epithelial program in our cell models, we
have (a) induced constitutive EMT in epithelial tumor subpopulations through the
transduction and overexpression of EMT-directing transcription factors, (b) knocked
down the same factors in the mesenchymal-like PC-3/S cell subpopulation, (c) knocked
down E-cadherin in the epithelial tumor subpopulations, (d) transduced and
overexpressed E-cadherin in PC-3/S cells, (e) knocked down self-renewal/pluripotency
factors in the strongly epithelial PC-3/Mc subpopulation, and (f) transduced and
overexpressed the self-renewal factor SOX2 and the epithelial gene
E-cadherin in the mesenchymal-like PC-3/S tumor cells. The results from all these
complementary approaches lead to the same conclusions, namely that the suppression
of an epithelial program (through constitutive expression of EMT transcription
factors or knockdown of E-cadherin) inhibits the self-renewal/pluripotency gene
network of tumor cells, their capacity to grow under attachment-independent
conditions, and their tumorigenic and metastatic potentials. The association between
properties attributed to TICs and an epithelial phenotype is further supported by
the fact that knockdown of 3 of the 4 canonical Yamanaka pluripotency transcription
factors (SOX2, KLF4, and MYC) in
PC-3/Mc cells reduced their epithelial phenotype and TIC attributes and induced an
invasive and more mesenchymal phenotype, while the overexpression of
SOX2 in PC-3/S cells was sufficient to enhance the expression
of epithelial markers and properties of TICs in these cells, including enhanced
tumorigenicity, while inhibiting invasiveness and the expression of mesenchymal
markers.

These results suggest that the self-renewal properties of these tumor cells depend on
the same factors that endow normal cells with self renewal and pluripotency (31, 32),
that this gene network sustains the expression of an epithelial gene program and, at
the same time, opposes the expression of a mesenchymal gene program and the
acquisition of a motile and invasive phenotype. Reciprocally, the induction of a
mesenchymal gene program in our cells opposes not only their epithelial gene
program, but also their self-renewal gene network and associated properties. This
situation is reminiscent of the requirement for normal adult fibroblasts to undergo
a mesenchymal-epithelial transition (MET) for their reprogramming into self-renewing
pluripotent cells (49–51). In fact, it has been shown that the
expression of E-cadherin by itself can facilitate the reprogramming of adult
fibroblasts and the acquisition of pluripotency (52, 53). Our results suggest that
the association between self renewal and epithelial gene programs also holds true
for at least the 2 cellular models studied here, derived from prostate and bladder
cancers.

The contraposition between a gene program that drives self renewal and an invasive
program also has precedents in other biological settings. During normal vertebrate
development, neural crest progenitor cell migration and specification require the
activation, among other factors, of Snail/Slug and concomitant suppression of Sox2,
events that are induced both by diffusible factors and cell-cell interactions (54, 55).
On the other hand, epithelial reprogramming is required for the induction or
maintenance of pluripotent states, which are facilitated by inhibition of EMT (49, 51),
while induction of EMT by SNAI1 can be antiproliferative (56, 57). This evidence
and our own observations suggest that mutual exclusion between progenitor/stem cell
(or CSC in tumors) and EMT programs may be the prevalent mode in the differentiation
of normal progenitor cells in some tissues and also in the evolution of some
epithelial tumors.

The suppression of major attributes of TICs by constitutive EMT found in our study
may seem to contradict other models, in which EMT induced by a number of factors
potentiates self renewal together with enhanced tumorigenic and metastatic
capacities (22, 24). However, our observations are supported by models in which
local invasiveness is inversely correlated with metastatic or organ colonization
potential (25, 28). Additional studies, including some in which EMT is
proposed to enhance tumorigenic and metastatic potentials, have also shown that
tumor cell subpopulations with clear epithelial phenotypes are endowed with the
strongest metastatic potential (23, 28, 58,
59). Our observations led us to propose
that tumor cells that depend on a self-renewal/pluripotency gene network for their
aggressive properties may be susceptible to an inhibition of those properties by
EMT, perhaps through direct or indirect downregulation of the
self-renewal/pluripotency gene network by factors such as SNAI1. We speculate that
tumor cell types that do not depend on the self-renewal/pluripotency gene network
for their tumorigenic and metastatic potentials may use EMT factors to induce a
self-renewal state, as described for several models (23, 60), through a variety of
mechanisms. Tissue-specific regulatory networks could also account for different
phenotypic consequences of EMT in different cell types. For example, it has been
reported that EMT of primary prostate epithelial cells is not accompanied with
enhanced anchorage-independent growth, (61),
and as we have found in this study, EMT can suppress the self-renewal states of
prostate and bladder cancer cells, while the induction of EMT in noncancerous MCF10
breast epithelial cells enhances their potential to form mammospheres (22, 24).

A second major finding of our study is that, during cooperation between epithelial
and mesenchymal-like tumor subpopulations, the former transiently undergo an EMT.
The cooperation between epithelial and mesenchymal-like tumor cell subpopulations
for enhanced local invasiveness has been described in other models (25, 26).
Our findings add a further level of regulatory complexity in these cell-cell
interactions and suggest that the escape of epithelial TICs from their primary sites
is facilitated by both passive and active mechanisms. Passive escape mechanisms
include the breakdown of extracellular matrix and other tissue structures by
tumor-associated stromal cells (8, 19, 48)
and by tumor cells that have acquired relatively stable mesenchymal-like gene
programs (25, 26). We consider here as an active mechanism the reversible acquisition
of a mesenchymal-like invasive state by epithelial tumor cells with self-renewal
potential, as observed in this study. In this scheme, it is important to distinguish
between tumor cell subpopulations that have acquired relatively stable
mesenchymal-like phenotypes and those subpopulations with strong epithelial
phenotypes that can undergo transient EMT. In our prostate cancer model, the former
subpopulation is unable to establish distant metastases, while the latter cells
maintain their capacity to metastasize, suggesting that they undergo a reversion of
the EMT, or MET, after they escape from their primary sites. The latter hypothesis
is supported by our observations that many experimental metastases express
E-cadherin and that metastases from prostate cancer patients also frequently display
a strong expression of E-cadherin.

Collectively, our observations suggest that, in some cancers, the acquisition of
mesenchymal traits by tumor cells that leads to their loss of epithelial properties
occurs at the expense of their self-renewal potential. When the induction of EMT is
constitutive, as by forced overexpression of Snai1, the losses in
self renewal and tumorigenic and metastatic potentials are also sustained. On the
other hand, transient EMT, such as that induced by the cooperation between
epithelial and mesenchymal tumor subpopulations described in this study, could
enhance the local invasiveness of the epithelial subpopulation, thus contributing to
the overall metastatic potential of a tumor in which heterogeneous epithelial and
mesenchymal subpopulations coexist. This model is schematically summarized in Figure
14 and proposes that the more
epithelial/self-renewal tumor populations that leave their primary site either
passively, aided by stromal or mesenchymal-like tumor cells, or actively, through
their own transient EMT, can form metastases because they have maintained their
epithelial phenotypes or, if they have undergone EMT at the primary site, revert to
an epithelial/self-renewal program at distant sites. We further propose that, once
at their metastatic sites, epithelial TICs may follow a cycle similar to that
occurring at the primary sites, with induction of EMT at varying degrees, depending
on the environment of the metastatic sites. Formal proof of this model, in
particular the demonstration of transient local EMT of epithelial TICs followed by
MET at distant sites, would require additional experimental confirmation. Our model
is compatible with a more direct participation of stromal or other nontumoral cells
in promoting the local invasiveness of tumor cells (8).

A model of metastasis potentiated by cooperation between tumor cell
populations expressing either epithelial/TIC or mesenchymal
programs. We propose a model in which some TICs, with properties of CSCs, undergo EMT
under the influence of environmental factors. This results in epigenetic
reprogramming, including a repression in those cells of pluripotency
programs that sustain cell self renewal. These
“mesenchymalized” cells, in turn, either through
direct cell-cell interaction or through diffusible factors, drive the
mesenchymal conversion of additional populations of TIC/CSCs that have not
yet undergone EMT, resulting in a reinforcement of the mesenchymalization of
the tumor. The predominantly mesenchymalized populations of tumor cells
complete the breach of local barriers and thus the tumor becomes fully
invasive. The tumor cells escaping from the local site would thus be a
combination of stably mesenchymalized tumor cells, cells retaining TIC/CSC
properties that leave the tumor following paths open by actively invading
cells (passive escape), or TIC/CSCs that have undergone transient EMT
(active escape). After hematogenous or lymphogenous spread, TIC/CSCs that
have not undergone EMT or that have reverted to an epithelial program and
phenotype from their transient EMT (MET) can establish distant metastases.
This cycle may be repeated at the metastatic site. Tumor cells with stable
mesenchymal-like phenotypes that have escaped from the local tumor site but
that do not revert to an epithelial gene program and phenotype would not
have the capacity to establish distant metastases.

Methods

Additional information appears in Supplemental Methods.

Cell lines and reagents. PC-3/Mc and PC-3/S were clonally derived from the human cell line PC-3, isolated
from the bone metastasis of a prostate adenocarcinoma (62). Both sublines carry the integrated firefly luciferase
gene coding region cloned in the Superluc pRC/CMV vector (Invitrogen). The
PC-3/Mc clone was selected by limiting dilution from PC-3/M, isolated from liver
metastases produced in nude mice subsequent to intrasplenic injection of PC-3
cells (30). PC-3/S cells were selected by
limiting dilution from parental PC-3 cells. Cells were grown at 37°C
in a 5% CO2 atmosphere in complete RMPI 1640 supplemented with 200
μg/ml Geneticin (Sigma-Aldrich) to maintain the chromosomal
integration of the luciferase gene. TSU-Pr1 and B2 cells (36, 37) were
maintained at 37°C in a 5% CO2 atmosphere in complete
DMEM. All media were supplemented with 2 mM l-glutamine, 100 U/ml
penicillin, 100 μg/ml streptomycin, and 10% FBS. Unless otherwise
indicated, media and sera were from PAA.

Coculture and cell-sorting experiments. PC-3/Mc cells were labeled with Oregon Green 488 carboxy-DFFDA-SE (Invitrogen)
with excitation maximum at 488 nm and emission at 524 nm. PC-3/S cells were
labeled with Far Red DDAO-SE (Invitrogen), with excitation maximum at 600 nm and
emission at 670 nm. Cells were labeled by adding 25 μM of
fluorophore to the cell suspensions for 30 minutes, washed with PBS, and
reseeded. Fluorophore-preloaded cells were cocultured at a 1:1 proportion for 48
or 96 hours and either assayed for invasiveness on Transwell-Matrigel chambers
or sorted with a FACSAria SORP instrument (BD). After sorting, cells were either
assayed for invasiveness in Transwell-Matrigel assays or processed for protein
extraction (for Western blotting), RNA extraction (for quantitative PCR [qPCR]),
or ChIP. As controls, PC-3/Mc cells were preloaded with Oregon Green, cocultured
with unlabeled PC-3/Mc cells, and sorted by FACS.

Invasiveness assays with PC-3/S CM. PC-3/S cells were cultured at 70% confluence, at which time they were changed to
fresh CD-CHO medium (Invitrogen) without FBS; CM was collected after 48 hours,
centrifuged, and filtered through a 0.22-μm filter (Millipore).
PC-3/Mc cells were cultured with PC-3/S CM (S-CM) mixed with fresh medium
without FBS at a 1.5:1 proportion for 48 hours, and these cells were then
analyzed for invasiveness and Western blotting.

Study approval. All tests in this study employing human tissues were performed on postdiagnosis
surplus samples obtained at the Hospital Clínic de Barcelona,
following protocols previously approved by the Hospital Clínic
Institutional Review Board. All animal studies were performed following
protocols previously approved by the CID-CSIC Institutional Review Board. As
independently certified by these 2 institutional review boards, all studies
involving human or animal materials or subjects were performed in compliance
with Spanish laws regulating ethics in research and patient data confidentiality
(Ley de Investigación Biomédica 14/2007, de 3 de Julio
de 2007).

Acknowledgments

We thank Dídac Domínguez and Marc Guiu (xenograft
experiments), Mònica Marín (immunohistochemistry),
Mònica Pons (confocal microscopy), and Jaume Comas and Ricard
ρlvarez (flow cytometry) for excellent technical assistance. T.
Celià-Terrassa is a recipient of a doctoral fellowship from the CSIC.
Ó. Meca-Cortés, A. Arnal-Estapé, and C.
Estarás are recipients of doctoral fellowships and F. Mateo of a Juan de
la Cierva postdoctoral fellowship from the Ministerio de Ciencia e
Innovación (MICINN). R.R. Gomis is a researcher of the
Institució Catalana de Recerca i Estudis Avançats. Financial
support was provided to T.M. Thomson by MICINN (SAF2008-04136-C02-01 and
SAF2011-24686), the Agència de Gestió d’Ajuts
Universitaris i de Recerca (2009SGR1482), the Agencia Española de
Cooperación Internacional y Desarrollo (A/023859/09), and the Xarxa de
Referencia en Biotecnologia; to P.L. Fernández by MICINN (FIS-PI080274),
the Fondo de Investigaciones de la Seguridad Social (PI080274), the Spanish National
Biobank Network, the Instituto de Salud Carlos III (ISCIII-RETIC RD06/0020), the
Xarxa de Bancs de Tumours de Catalunya-Pla Director d’Oncologia, and the
Fondo Europeo de Desarrollo Regional (FEDER) — Unión Europea
“Una manera de hacer Europa”; and to R.R. Gomis by the BBVA
Foundation and MICINN (SAF2007-62691).

Footnotes

Conflict of interest: The authors have declared that no conflict of
interest exists.